1. Field of the Invention
The present invention relates to peptide analogs and peptide mimetics of xcex2-sheet breaker peptides suitable for in vivo use in treating mammals with protein conformational diseases such as Alzheimer""s and prion disease. More particularly, the present invention is directed to novel peptide analogs and mimetics, pharmaceutical compositions containing one or a mixture of such peptide analogs and mimetics, and methods for preventing, treating, or detecting disorders or diseases associated with abnormal protein folding into amyloid or amyloid-like deposits or precursors thereof having a pathological beta-sheet structure.
2. Description of Related Art
Extensive evidence has been accumulated indicating that several diverse disorders have the same molecular basis, i.e. a change in a protein conformation (Thomas et al., Trends Biochem. Sci. 20: 456-459, 1995; Soto, J. Mol. Med. 77: 412-418, 1999). These protein conformational diseases include Alzheimer""s disease, prion-related disorders, systemic amyloidosis, serpin-deficiency disorders, Huntington""s disease and Amyotrophic Lateral Sclerosis (Soto 1999, supra). The hallmark event in protein conformational disorders is a change in the secondary and tertiary structure of a normal protein without alteration of the primary structure. The conformationally modified protein may be implicated in the disease by direct toxic activity, by the lack of biological function of normally-folded protein, or by improper trafficking (Thomas et al., 1995, supra). In the cases where the protein is toxic, it usually self-associates and becomes deposited as amyloid fibrils in diverse organs, inducing tissue damage (Thomas et al., 1995, supra; Kelly, Curr. Opin. Struct. Biol. 6: 11-17, 1996; Soto, 1999, supra).
Alzheimer""s disease (AD) is a devastating neurodegenerative problem characterized by loss of short-term memory, disorientation, and impairment of judgment and reasoning. AD is the most common dementia in elderly population. It is estimated that more than twenty-five million people worldwide are affected in some degree by AD (Teplow, Amyloid 5: 121-142, 1998). A hallmark event in AD is the deposition of insoluble protein aggregates, known as amyloid, in brain parenchyma and cerebral vessel walls. The main component of amyloid is a 4.3 KDa hydrophobic peptide, named amyloid beta-peptide (Axcex2) that is encoded on the chromosome 21 as part of a much longer precursor protein (APP) (Selkoe, Science 275: 630-631, 1997). Genetic, biochemical, and neuropathological evidence accumulated in the last 10 years strongly suggest that amyloid plays an important role in early pathogenesis of AD and perhaps triggers the disease (Soto et al., J. Neurochem. 63: 1191-1198, 1994; Selkoe, 1997, supra; Teplow, 1998, supra; Sisodia and Price, FASEB J. 9: 366-370, 1995; Soto, Mol. Med. Today 5: 343-350, 1999).
Amyloid is a generic term that describes fibrillar aggregates that have a common structural motif, i.e., the xcex2-pleated sheet conformation (Serpell et al., Cell Mol. Life Sci. 53: 887, 1997; Sipe, Ann. Rev. Biochem. 61: 947-975, 1992). These aggregates exhibit specific tinctorial properties, including the ability to emit a green birefringent glow after staining with Congo red, and the capacity to bind the fluorochrome, thioflavin S (Sipe, 1992, supra; Ghiso et al., Mol. Neurobiol. 8: 49-64, 1994). There are more than a dozen human diseases of different etiology characterized by the extracellular deposition of amyloid in diverse tissues, which lead to cell damage, organ dysfunction, and death. Among the diseases involving amyloidosis, it is possible to highlight Alzheimer""s disease, prion-related disorders (also known as transmissible spongiform encephalopathy), and systemic amyloidosis (Table 1). The amyloid fibrils are usually composed of proteolytic fragments of normal or mutant gene products. There are over 16 different proteins (Table 1) involved in amyloid deposition in distinct tissues (Ghiso et al., 1994, supra).
The formation of amyloid is basically a problem of protein folding, whereby a mainly random coil soluble peptide becomes aggregated, adopting a xcex2-pleated sheet conformation (Kelly, 1996, supra; Soto, 1999, supra). Amyloid formation proceeds by hydrophobic interactions among conformationally altered amyloidogenic intermediates, which become structurally organized into a xcex2-sheet conformation upon peptide interaction. The hydrophobicity appears to be important to induce interaction of the monomers leading to aggregation, while the xcex2-sheet conformation might determine the ordering of the aggregates in amyloid fibrils. In an attempt to inhibit amyloid fibril formation, these two properties were separated by designing short synthetic peptides bearing sequence homology and a similar degree of hydrophobicity as the peptide domain implicated in the conformational change, but having a very low propensity to adopt a xcex2-sheet conformation (called xcex2-sheet breaker peptides) (Soto et al., 1996, supra; Soto et al., 1998, supra). The aim was to design a peptide with the ability to bind specifically to the amyloidogenic peptide forming a complex that stabilizes the physiological conformation and destabilizes the abnormal conformation of the peptide (Soto, 1999, supra).
xcex2-sheet breaker peptides have so far been designed to block the conformational changes that occur in both Axcex2 and prion protein (PrP), which are implicated in the pathogenesis of Alzheimer""s and prion disease, respectively. The prior art has previously shown that 11- and 5-residue xcex2-sheet breaker peptides (namely, iAxcex21 and iAxcex25, respectively) homologous to the central hydrophobic region of Axcex2 inhibit peptide conformational changes that result in amyloid formation and also dissolved preformed fibrils in vitro (Soto et al., Biochem. Biophys. Res. Commun. 226: 672-680, 1996; Soto et al., Nature Med. 4: 822-826, 1998). In addition, the 5-residue peptide is capable of preventing the neuronal death induced by the formation of xcex2-sheet rich oligomeric Axcex2 structures in cell culture experiments (Soto et al., 1998, supra). Furthermore, by using a rat model of amyloidosis induced by intracerebral injection of Axcex21-42, the prior art has shown that co-injections of the 5-residue xcex2-sheet breaker peptide decreased cerebral Axcex2 accumulation and completely blocked the deposition of fibrillar amyloid-like lesions in the rat brain (Soto et al., 1998, supra). Finally, the xcex2-sheet breaker peptide injected eight days after the injection of Axcex2 was able to disassemble preformed Axcex2 fibrils in the rat brain in vivo, that leads to a reduction in the size of amyloid deposits (Sigurdsson, E. M.; Permanne, B.; Soto, C.; Wisniewski, T.; Frangione, B.; J. Neuropathol. Exp. Neurol. January 2000; 59(1): 11-7). Interestingly, removal of amyloid by the xcex2-sheet breaker peptide reverts the associated cerebral histologic damage, including neuronal shrinkage and microglial activation.
xcex2-sheet breaker peptides have also been designed to prevent and to revert conformational changes caused by prions (PrP). Based on the same principles and using as a template the PrP sequence 114-122, the prior art has shown that when a set of xcex2-sheet breaker peptides was synthesized, a 13-residue peptide (iPrP13) showed the greatest activity (Soto, 1999, supra). Several in vitro cell culture and in vivo assays were used to test for inhibitory activity and the results clearly indicated that it is possible not only to prevent the PrPcxe2x86x92PrPsc conversion, but more interestingly to revert the infectious PrPsc conformer to a biochemical and structural state similar to PrPc (Soto et al., manuscript submitted).
Short peptides have been utilized extensively as drugs in medicine (Rao et al., C. Basava and G. M. Anantharamaiah, eds. Boston: Birkhauser, pp. 181-198, 1994). However, the development of peptide drugs is strongly limited by their lack of oral bioavailability and their short duration of action resulting from enzymatic degradation in vivo (Fauchere and Thurieau, Adv. Drug Res. 23: 127-159, 1992). Progress in recent years toward the production of peptide analogs (such as pseudopeptides and peptide mimetics) with lower susceptibility to proteolysis has increased the probability to obtain useful drugs structurally related to their parent peptides (Fauchere and Thurieau, 1992, supra). Improving peptide stability to proteases not only increases the half-life of the compound in the circulation but also enhances its ability to be transported or absorbed at different levels, including intestinal absorption and blood-brain barrier permeability, because transport and absorption appear to be highly dependent upon the time of exposure of membranes or barriers to the bioactive species (Fauchere and Thurieau, 1992, supra).
The present invention is an inhibitory peptide capable of inhibiting xcex2 pleated sheet formation in amyloid xcex2-peptide, the inhibitory peptide being a xcex2sheet breaker peptide analog designed by chemical modification of a xcex2sheet breaker peptide capable of inhibiting xcex2 pleated sheet formation in amyloid xcex2-peptide.
The peptide is altered chemically by: (1) modifications to the N- and C-terminal ends of the peptide; (2) changes of the side-chain, which can involve amino acid substitutions; (3) modification in the xcex1-carbon including methylations, alkylations and dehydrogenations; (4) chirality changes by replacing D- for L-residue; (5) head-to-tail cyclizations; and (6) introduction of amide bond replacements, i.e. changing the atoms participating in the peptide (or amide) bond.
The present invention also includes an inhibitory peptide capable of inhibiting conformational changes in prion PrP protein associated with amyloidosis, the inhibitory peptide being a xcex2sheet breaker peptide analog designed by chemical modification of a xcex2sheet breaker peptide capable inhibiting the conformational changes in prior PrP protein associated with amyloidosis.
In addition, the present invention includes a peptide mimetic with the following structure: 
In another embodiment, the peptide mimetic has the following structure: 
In yet another embodiment, the peptide mimetic has the following structure: 
The present invention also includes a method for preventing, treating, or detecting disorders or diseases associated with abnormal protein folding into amyloid or amyloid-like deposits or precursors thereof having a pathological beta-sheet structure is claimed: